Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome

To investigate the effect of bromodomain and extra-terminal (BET) protein inhibitor JQ1 on expression of autoimmune-related genes in CD4+T cells from patients with systemic lupus erythematosus (SLE).
 Methods: Peripheral CD4+T cells were isolated by positive selection with CD4 microbeads. The percentage of CD4+T cells were detected by flow cytometry. CD4+T cells were treated by JQ1 at 100 nm/L for 6, 24, 48 h. The expression of T cell-related genes was measured by quantitative real-time PCR (qPCR). The secretion levels of cytokines in culture supernatant were measured by ELISA at 48 h.
 Results: The percentage of CD4+T cells isolated by CD4 microbeads is 97.2%. Compared with the control group, the mRNA expression levels of IFNG, IL-17F, IL-21, CXCR5 and FOXP3 were down-regulated at 6, 24 and 48 h (P<0.05), and IL-17A mRNA level was decreased at 6 and 24 h (P<0.01); while IL-4 mRNA level was up-regulated at 24, 48 h (P<0.01), and TGF-β1 mRNA level was up-regulated at 6 and 48 h (P<0.05) in SLE CD4+T cells treated with JQ1. The secretion levels of IFN-γ and IL-21 in JQ1-treated group were decreased significantly (P<0.05), while the secretion levels of IL-4 and TGF-β were up-regulated compared with control group (P<0.05).
 Conclusion: JQ1 can reverse the immune dysregulation and improve the immunity homeostasis in CD4+T cells from patients with SLE.
Azepines ; pharmacology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; metabolism ; Cytokines ; analysis ; metabolism ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Proteins ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Time Factors ; Transforming Growth Factor beta1 ; Triazoles ; pharmacology

To explore the role of methotrexate (MTX) in regulating the number of regulatory T cells (Treg) and the mRNA expression of transcription factor Foxp3.
 Methods: 1) We analyzed the number of Treg and the mRNA expression of Foxp3 by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR) respectively in patients with psoriasis vulgaris, patients with psoriasis vulgaris after the 8-week treatment of MTX, and healthy people. 2) BALB/c female mice were smeared with imiquimod (IMQ) cream for 6 days. We recorded the change of the lesion in mice every day. The morphological changes of lesion in mice were evaluated by the psoriasis area and severity index (PASI) and HE staining. 3) The mouse model was randomly divided into a control group and an MTX group. The MTX group was treated with different doses of MTX (38.5 and 77.0 nmol/L) on the third day of this experiment. The morphological changes of lesion in mice were evaluated by PASI and HE staining. We tested the number of Treg and the expression level of Foxp3 mRNA in splenic lymphocytes.
 Results: 1) The number of Treg and the expression level of Foxp3 mRNA were lower in psoriasis vulgaris patients than those in the healthy control group (P<0.05). After 8-week treatment of MTX, the number of Treg was increased (P<0.05) and Foxp3 mRNA level was up-regulated (P<0.01). 2) Typical psoriasis-like skin lesions, such as red scaly skin plaque were found after topical application of IMQ. Both the number of Treg in the splenic lymphocytes of mice and the Foxp3 mRNA level of Treg were reduced by IMQ (P<0.01 and P<0.05). 3) Different doses of MTX for mice showed the ability to improve skin lesion, increase the number of Treg in the spleen of mice and Foxp3 mRNA level in psoriatic dermatitis of mice (P<0.05).
 Conclusion: MTX is able to regulate the number of Treg and Foxp3 mRNA expression in psoriasis.
Adjuvants, Immunologic ; pharmacology ; Aminoquinolines ; pharmacology ; Animals ; Case-Control Studies ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Imiquimod ; Immunosuppressive Agents ; administration & dosage ; pharmacology ; Lymphocyte Count ; Methotrexate ; administration & dosage ; pharmacology ; Mice ; Mice, Inbred BALB C ; Psoriasis ; drug therapy ; immunology ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; metabolism

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or "on-target/off-tumor" toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strategies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vector designs to increase both the safety and efficacy, further T cell modification to overcome the tumor-associated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal-curing cancer with high safety, high efficacy, and low cost.
Cell Movement ; immunology ; Cell Proliferation ; Gene Expression ; Genetic Vectors ; chemistry ; metabolism ; Humans ; Immunotherapy, Adoptive ; methods ; Lymphocyte Activation ; Lymphocytes, Tumor-Infiltrating ; cytology ; immunology ; transplantation ; Neoplasms ; genetics ; immunology ; pathology ; therapy ; Patient Safety ; Receptors, Antigen, T-Cell ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Signal Transduction ; Single-Chain Antibodies ; chemistry ; genetics ; T-Lymphocytes ; cytology ; immunology ; transplantation ; Treatment Outcome

OBJECTIVETo explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP).

METHODSThe ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR.

RESULTSThe ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-β and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls.

CONCLUSIONThe alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.

Animals ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; pathology ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Smad7 Protein ; metabolism ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; Transforming Growth Factor beta ; metabolism

BACKGROUNDAspergillosis infection is common in the patients with insufficient immunity. The role of mammalian target of rapamycin (mTOR), T-box expressed in T-cells (T-bet), and eomesodermin (EOMES) in mediating T lymphocytes differentiation in response to Aspergillus fumigatus infection in immunocompromised rats was investigated in this study.

METHODSInvasive pulmonary aspergillosis (IPA) of immunosuppressive twenty male rats were established and sacrificed at 24 h (n = 5), 48 h (n = 5), 72 h (n = 5), and 96 h (n = 5) after A. fumigatus infection. In addition, control (n = 5), cyclophosphamide (CTX) (n = 5), and aspergillosis (n = 5) group were also established the tissues and pathology of lung tissue was examined by hematoxylin and eosin staining. CD8+ T-cells was sorted by flow cytometry. Serum mTOR, S6K, T-bet, and EOMES were quantified by enzyme-linked immunosorbent assay.

RESULTSHistology of lung tissue indicated severe lung tissue injury including infiltration of inflammatory cells, alveolar wall damage or degradation, blood congestion, and hemorrhage in the CTX, IPA, and CTX + IPA rats. Hyphae were seen in the IPA, and CTX + IPA groups. The proportion of CD8+ T-cells was significantly increased in the animals of CTX + IPA. Memory CD8+ T-cells was significantly increased in early stage (24 h and 48 h, P < 0.001), but decreased in the late phase of fungal infection (72 h and 96 h) in the animals of CTX + IPA. In addition, at early stage of fungal infection (24 h and 48 h), serum mTOR (P < 0.001), S6K (P < 0.001), and T-bet (P < 0.05) was significantly higher, while EOMES was significantly lower (P < 0.001), in CTX + IPA group than that in control, CTX alone or IPA alone group. Conversely, serum mTOR, S6K, T-bet, and EOMES showed opposite changed in the late stage (72 h and 96 h). Pearson's correlation analysis indicated that mTOR and S6K were significantly correlated with T-bet (r = 0.901 and 0.91, respectively, P < 0.001), but negatively and significantly correlated with EOMES (r = -0.758 and -0.751, respectively, P < 0.001).

CONCLUSIONSmTOR may regulate transcription factors of EOMES and T-bet, and by which mechanism, it may modulate lymphocytes differentiation in animals with immune suppression and fungal infection.

Animals ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Invasive Pulmonary Aspergillosis ; metabolism ; pathology ; Lung ; metabolism ; pathology ; Lymphocytes ; cytology ; immunology ; Male ; Rats ; Rats, Wistar ; T-Box Domain Proteins ; genetics ; metabolism ; TOR Serine-Threonine Kinases ; genetics ; metabolism ; Tissue Culture Techniques

BACKGROUNDPostresuscitation immune dysfunction contributes to the low survival rate after successful resuscitation, but its mechanism remains poorly understood. The purpose of this study was to investigate whether splenic regulatory T-cell (Treg) apoptosis was involved in the postresuscitation immune dysfunction.

METHODSThirty-eight pigs were randomly divided into sham-operated group (SHAM group, n = 8), 12 h post return of spontaneous circulation (ROSC) group, 24 h post-ROSC group, and 48 h post-ROSC group (n = 10 per group). A Wuzhishan miniature porcine model of 8-min ventricular fibrillation cardiac arrest (CA) was established. The apoptosis rates of Treg in the spleen were tested by flow cytometry; the expressions of forkhead/winged helix transcription factor (Foxp3) of Treg in the spleen were detected by real-time polymerase chain reaction; and the levels of interleukin-4 (IL-4), IL-10, and interferon gamma (IFN-γ) of Treg in the spleen were detected by enzyme-linked immunosorbent assay.

RESULTSThe apoptosis rates of Treg in all post-ROSC groups were significantly lower than that of SHAM group (7.7% ± 1.9%, 7.1% ± 1.8%, 6.2% ± 0.4% vs. 13.1% ± 1.6%; P < 0.05); the expression levels of Foxp3 and IL-10 were also decreased with the increase of apoptosis rates of Treg. Helper T-cells CD4+ lymphocyte subsets were significantly lower in the post-ROSC groups compared with SHAM group (29.1% ± 2.2%, 24.3% ± 2.2%, 24.1% ± 2.5% vs. 43.8% ± 4.5%; P < 0.01) at 12, 24, and 48 h after ROSC. Compared with SHAM group, the levels of IFN-γ (161.0 ± 12.9, 167.7 ± 10.5, 191.2 ± 7.7 vs. 7.6 ± 0.9 ng/L) and IL-4 (27.7 ± 6.2, 35.9 ± 3.5, 50.6 ± 6.1 vs. 13.3 ± 2.3 ng/L) and the ratio of IFN-γ/IL-4 (8.6 ± 2.3, 4.9 ± 0.4, 4.5 ± 0.9 vs. 0.8 ± 0.2) were all greatly elevated in all post-ROSC groups (P < 0.05).

CONCLUSIONSApoptosis rate of Treg was significantly decreased after CA, and thus the proportion of Treg was increased and the inhibitory effects were enhanced, which further led to the decrease of the amount of CD4+ T-cells. In addition, the T helper type 2/T helper type 1 (Th2/Th1) cell drift of Treg in the spleen caused postresuscitation immune dysfunction.

Animals ; Apoptosis ; physiology ; Cardiopulmonary Resuscitation ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Heart Arrest ; immunology ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Random Allocation ; Spleen ; cytology ; Swine ; Swine, Miniature ; T-Lymphocytes, Regulatory ; cytology ; metabolism ; physiology ; Ventricular Fibrillation ; complications ; metabolism

OBJECTIVETo investigate the effect of tumor necrosis factor-α-induced protein 8 like-2 (TIPE2) on apoptosis of CD4T lymphocytes in a murine model of severe burn injury.

METHODSA total of 140 male mice were randomly allocated into 6 groups. Small RNA interference technique was used to construct a siTIPE2-overexpressing lentivirus, and severe burn injury models were established in the mice. CD4T cells were purified from spleen of the mice, and the expressions of TIPE2, Smad2/Smad3, P-Smad2/P-Smad3 and Bcl-2/Bimprotein in CD4Tregs were detected. The changes in mitochondrial membrane potential and cytochrome C in CD4T cells were detected, and the activities of caspase-3, caspase-8, and caspase-9 were analyzed.

RESULTSDown-regulation of TIPE2 promoted the apoptosis of CD4T lymphocytes in siTIPE2-burn group, in which the protein expressions of P-smad2/P-Smad3 decreased, Bcl-2 expression increased and Bim expression decreased significantly as compared with the other groups (P<0.01 or 0.05). The mitochondrial membrane potential and cytochrome C expression in CD4T cells were down-regulated in siTIPE2-burn group (P<0.05) with a lowered caspase-3 activity compared with TIPE2-burn group (P<0.01) and decreased caspase-8 and caspase-9 compared with the other groups (P<0.05). The apoptosis rate was the highest in TIPE2-burn group, whose Smad2/Smad3 was higher than that in the sham group (P<0.05) and the expression of P-smad2/P-Smad3 significantly increased compared with the other groups (P<0.05). In TIPE2-burn group, the mitochondrial membrane potential in CD4T cells was decreased (P<0.01), the expression of cytochrome C increased markedly (P<0.01), and the activities of caspase-3, caspase-8, and caspase-9 were all obviously higher than those in the other groups (P<0.05).

CONCLUSIONAs an important immunoregulatory molecule, TIPE2 can promote the apoptosis of CD4T lymphocyte in mice with sever burn injury.

Animals ; Apoptosis ; Burns ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Down-Regulation ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Spleen